The major objective of this research is to elucidate the chemical and physical properties of soluble methane monooxygenase (sMMO), a system of three proteins used by methanotrophic bacteria to convert methane and oxygen selectively to methanol and water. Methanotrophs consume significant amounts of methane, a greenhouse gas and their sole source of carbon and energy. These organisms are used in bioremediation of the environment, for example, to remove chlorinated hydrocarbons from drinking water supplies. Understanding the principles by which the enzyme system hydroxylates methane can provide key insights into the development of synthetic catalysts for achieving this important industrial goal. A principle component of sMMO is the hydroxylase enzyme (MMOH), which house two non-heme, carboxylate-bridged diiron centers where reductive activation of dioxygen takes places, evolving species that ultimately oxidize methane. Related chemistry occurs at similar cores located in the small subunit of ribonucleotide reductase (RNR), an enzyme which catalyzes the first step in DNA biosynthesis and which is a target of anti-tumor and anti-viral agents. Among the specific aims of this project is to understand the details of how these non-heme iron centers achieve such remarkable transformations under physiological conditions. Advanced methodologies will be applied to trap and determine the structures of intermediates in the MMOH reaction cycle, including rapid freeze-quench EPR and ENDOR spectroscopic. and double-mixing stopped-flow experiments, and to examine the reactivity of time-resolved intermediates with substrates. In parallel with work on the enzyme, synthetic analogs of the carboxylate-bridged diiron cores of MMOH and RNR will be prepared to help understand their active sites and to reproduce steps in their catalytic cycles. Kinetic and mechanistic experiments will be performed the learn the factors which control alkane hydroxylation, alkene epoxidation, tyrosyl radical generation, oxidase and peroxidase activities of the bridged diiron centers. The other two components of the sMMO system, a reductase (MMOR) and a small coupling protein (MMOB), form complexes with MMOH and significantly alter its catalytic activity and redox properties. Additional goals are to determine the structures of both these proteins by NMR and X-ray diffraction methods and to investigate the formation of complexes between all three components by thermodynamic and kinetic measurements. The optical spectra of the flavin and [2Fe-2S] chromophores in MMOR will be used to track electron-transfer reactions through the system. Site-directed mutagenesis studies of all three proteins will be carried out to identify key amino acid residues postulated to be involved in complex formation, electron transfer, proton transfer, substrate access to the diiron center, and the hydroxylation chemistry.